She was appointed Parliamentary Secretary to the Minister for Defence in July 2004, and Parliamentary Secretary to the Minister for Foreign Affairs and Trade in January 2006. In 2007 she was appointed Assistant Minister for Immigration and Citizenship.[2]

She re-entered Federal Parliament as the member for Brisbane at the 2010 federal election, becoming the first woman to represent Brisbane since its creation in 1901. Gambaro ran under the banner of the Liberal National Party, formed in 2008 after the merger of the Queensland divisions of the Liberal and National parties. She sits with the Liberals.

In September 2010 she was appointed Shadow Parliamentary Secretary for International Development Assistance and Shadow Parliamentary Secretary for Citizenship and Settlement by Opposition leader, Tony Abbott.[3]

Gambaro made news in January 2012 by suggesting that immigrants are not integrating into Australian culture well enough. She believes that telling them to "wear deodorant" and learning to "wait in a queue" is "teaching what are norms in Australia." After a storm of outcry on social media and in Australian newspapers, Gambaro apologised.[4][5]

In August 2013 Gambaro announced her support for same-sex marriage, despite party policy being against it, and said she had pressed Abbott for a conscience vote on the issue.[6]